Anti α tubulin

HeLa cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min, washed three times with PBS. Nonspecific binding sites were blocked with 10% normal goat serum, 0.1% Triton X-100 for 1 h at room temperature. The cells were then stained for 2 h at room temperature with the primary binder / antibody. After that, the cells were washed three times with PBS containing 0.1% TritonX-100, following by staining for 1 h with the secondary antibody. Finally, cells were washed three times with PBS containing 0.1% Triton X-100, and mounted with ProLong Gold Antifade reagent with DAPI (Invitrogen). Alexa Fluor 488 or 647 conjugated F(ab')₂ fragment goat anti-human IgG, F(ab')2 fragment specific (Jackson ImmunoResearch) was used as the secondary antibody (1:500) when using sAB-K29 as the primary binder (2 μg/mL). Other primary antibodies are diluted as followed: Anti AuroraB (Fisher,1:500); Anti α-Tubulin (Genscript, 1:50-1:100); Anti ɑ-Tubulin (proteintech, 1:200); Anti c-Myc (Genscript, 0.5 μg/mL); Anti EIF3B (Santa Cruz, 1:100); Anti INCENP (Santa Cruz,1:100), Anti MKLP1(Santa Cruz, 1:100); Anti Proteasome 20S core subunits (Fisher, 1:250); Anti K48-Ub (Abcam, 1:200); Anti VCP (Santa Cruz,1:200); Anti G3BP1 (proteintech,1:500); Anti pTBK1 (cell signaling,1:50); Anti PLK1 (Santa Cruz,1:50). Images were collected by a Leica SP5 2photon laser scanning confocal microscope.